Porcine stem cell factor varients and recombinant cells expressing such polypeptides

ABSTRACT

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been identified as porcine stem cell factors, and in particular membrane-bound porcine stem cell factors, and still more particularly as being involved in the culture of pluripotent or totipotent porcine cells.

This application claims the benefit of priority of U.S. ProvisionalApplication Serial No. 60/055,735, filed Aug. 13. 1997.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production and isolation of suchpolynucleotides and polypeptides. More particularly, the polynucleotidesand polypeptides of the present invention have been identified as beingvariant porcine stem cell factors, and in particular membrane-bound stemcell factors, and still more particularly as being involved in stemcell, useful for supporting proliferation and growth of porcine bonemarrow cells.

SUMMARY OF THE INVENTION

The present invention provides in one aspect a novel polypeptide whichhas been characterized as an active form of porcine stem cell factorcDNA gene sequence(PSCF) that omits exon 6. Preferably, the PSCF is asplice variant wherein exon 6 is omitted. More preferably, exon 6 isremoved from the native full-length porcine stem cell factor and isomitted entirely or is replaced with a polynucleotide that encodes oneor more amino acids.

In a preferred aspect the invention provides a PSCF encoded by apolynucleotide sequence corresponding to the full-length native porcinestem cell factor cDNA, but in which: (1) the first 70 nucleotides areremoved, (2) exon 6 is excised (nucleotides 591 to 654) from the fullpolynucleotide sequence, (3) the excised exon 6 segment is replaced by athree-nucleotide segment encoding the amino acid “Gly”, and (4) thefifteen-nucleotide C-terminal tail (nucleotides 938-952) is removed andreplaced by the six-nucleotide segment 5′-TCTAGA-3′ (SEQ ID NO: 11).

In accordance with another aspect of the present invention, there areprovided novel PSCF polypeptides, as well as active fragments, analogsand derivatives thereof. In a preferred aspect the present inventionprovides novel membrane-bound PSCF polypeptides, wherein the polypeptidesegment encoded by exon 6 is omitted or replaced by an inactivepolypeptide segment. In another preferred aspect the present inventionprovides such novel PSCF polypeptides which are soluble PSCFpolypeptides.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding the polypeptides ofthe present invention including mRNAs, cDNAs, genomic DNAs as well asactive analogs and fragments of such polypeptides.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence of the presentinvention, under conditions promoting expression of said polypeptides.

In accordance with a further aspect of the invention, the polypeptide ofthe invention may be anchored to a cell's surface, and the modifiedcell, as a feeder cell for culturing porcine cells.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides for analyzingpotential agonists to the polypeptides, Another process utilizes thepolynucleotides to assay for compounds which bind said polynucleotidesand would thus block expression of any products from saidpolynucleotides.

In accordance with yet a further aspect of the present invention, thereare also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for purposes related toscientific research for example, to generate probes for identifyingsimilar sequences which might encode similar polypeptides from otherorganisms by using certain regions, i.e., conserved sequence regions, ofthe nucleotide sequence.

In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode PSCFpolypeptides, in which polynucleotides exon 6 has been removed,deactivated or replaced by an inactive portion (SEQ ID NO:8) as shown inFIG. 4, as well as said encoded mature PSCF polypeptide as shown in FIG.4 (SEQ ID NO:9).

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

Definitions

In order to facilitate understanding of the following description andexamples which follow certain frequently occurring methods and/or termswill be described.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening non-codingsequences (introns) between individual coding segments (exons).

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequencesultimately process to produce the desired polypeptide.

“Recombinant” polypeptides refer to polypeptides produced by recombinantDNA techniques; i. e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. “Synthetic” polypeptidesare those prepared by chemical synthesis.

A DNA “coding sequence of” or a“nucleotide sequence encoding” aparticular polypeptide, is a DNA sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate regulatory sequences.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel or an agarose gel to isolate thedesired fragment.

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate group with an ATP in the presence of akinase. A synthetic oligonucleotide will ligate to a fragment that hasnot been dephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (J. Sambrook et al., 1989, inMolecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory,NY). Unless otherwise provided, ligation may be accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, 1989.

“Porcine Stem Cell Factor” (PSCF) or “porcine steel factor” are termsthat are used herein interchangeably. Also the corresponding mousefactors are discussed as being “mouse stem factor” (MSF) or “mouse steelfactor”. PSCF is also called factor, porcine mast cell growth factor andporcine c-kit ligand in the art. Each of the native steel factors (SFs)has a transmembrane polypeptide with a cytoplasmic domain and anextracellular domain. Soluble PSCF or MSF refer to a fragment cleavedfrom the extracellular domain at a specific proteolytic cleavage site(e.g. for soluble PSCF, amino acids 1-160. Membrane associated SF refersto both normal SF before it has been cleaved or the SF which has beenaltered so that proteolytic cleavage cannot take place. The PSCF may beeither a soluble form as described in U.S. Pat. No. 5,589,582, or itsequivalents, or may be a membrane-bound form, preferably bound on to thesurface of a cell.

“Feeder cells” are cells which produce membrane-bound and/or solublePSCF, preferably a fibroblast cell that is transformed to produce thePSCF, more preferably are transformed murine fibroblast cells, and evenmore preferably are murine fibroblast cells known as the STO cell linethat have been transformed. Particularly, preferred feeder cells havethe polypeptide according to the invention anchored to the surface ofthe cell. Such feeder cell lines may be referred to above andhereinafter as “STO5”, “STO8”, “STO12” or “STO18” cells. Feeder cellswhich produce murine stem cell factor MSCF may be referred to as STOcells (the STO cell line is a thioguanine/oubain resistant sub-line ofSIM mouse fibroblasts, Virology 50:339 (1972); STO cells are describedin U.S. Pat. No. 5,453,357). The full-length amino acid sequence forPSCF (SEQ ID NO:2) based on native cDNA (SEQ ID NO:1 and FIG. 1 attachedhereto) is reported in Biology of Reproduction 50: 95-102 (1994), andactive forms can be produced by transfecting cells with active fragmentsof the cDNA sequence which may have the polynucleotides encoding theleader sequence amino acids (−25 to −1) removed. Soluble formspreferably omit the polynucleotide transmembrane portion. Additionally,an active form of PSCF (as in the present invention) can be produced andutilized to transfect STO cells by removing exon 6 from the full lengthcDNA and substituting a polynucleotide segment that encodes one or moreamino acids. Active forms of the PSCF of the present invention may alsoomit the C-terminal polypeptide segment corresponding to nativepolypeptide beginning with amino acid 217 (see, FIG. 1 or SEQ ID NO:2for such C-terminal segment).

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of an embodiment of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1 illustrates the full-length sequence for PSCF (SEQ ID NO:2) basedon native cDNA (SEQ ID NO:1) as reported in Biology of Reproduction 50:95-102 (1994).

FIG. 2 illustrates the polynucleotide sequence (SEQ ID NO: 5) encoding asoluble form of PSCF (SEQ ID NO: 10) from U.S. Pat. No. 5,589,582.

FIG. 3 shows a comparison of proliferation results for pig bone marrowcells cultured in the presence of STO cells as compared to STO cellstransfected with the polynucleotide sequence that is set forth in FIG.4. The proliferation results for pig bone marrow cells cultured in thepresence of the transfected cells are significantly better than resultswith regular STO cells.

FIG. 4 illustrates the polynucleotide sequence (SEQ ID NO:8) encoding anactive membrane-bound form of PSCF (SEQ ID NO:9) from which exon 6 hasbeen been removed and replaced with a tri-nucleotide that encodes theamino acid “Gly” which is part of plasmid pPSCF.

DETAILED DESCRIPTION OF THE INVENTION

The polynucleotides of this invention coding for the polypeptides ofthis invention were originally recovered from a pig bone marrow stromalcells and modified to remove exon 6 as well as the C-terminal portion ofthe gene. Exon 6 was replaced with a tri-nucleotide “GGG” which encodesa “Gly” amino acid, but any polynucleotide encoding a polynucleotidesequence in phase with the remaining coding portion of the C-terminalportion of the PSCF native gene, which does not inactivate the resultingPSCF polypeptide may be used instead of the GGG tri-nucleotide.

The STO5, STO8, STO12 and STO18 are feeder cells according to thepresent invention wherein STO cells have been transfected with apolynucleotide which encodes a membrane-bound portion (or portions) ofan active PSCF polypeptide.

As described above, to express the membrane form of porcine stem cellfactor (PSCF) in the mouse fetal fibroblast feeder cell line STO and toprovide STO5, STO8, STO12 and STO18 cell lines or the equivalent, theSTO cells may be transfected with a portion of the cDNA encoding thePSCF gene. Alternatively, by eliminating the membrane-binding portion(portion corresponding to nucleotides 715 to 783 of the full length cDNAas shown in FIG. 1), the soluble form of PSCF may be produced by STOcells or the like transfected with the polynucleotide encoding thesoluble PSCF polypeptide.

For example, STO8 cells are produced by transfecting STO cells with apolynucleotide sequence corresponding to the full-length cDNA in which:(1) the first 69 polynucleotides are removed, (2) exon 6 is excised(polynucleotides 592 to 654) from the full polynucleotide sequence, (3)the excised exon 6 segment is replaced by a three-nucleotide segmentencoding the amino acid “Gly”, and (4) the fifteen-nucleotide C-terminaltail (polynucleotides 938-952) is removed and replaced by thesix-polynucleotide segment 5′-TCTAGA-3′.

Other active PSCF polynucleotides may be utilized. Particularlyperferred are polynucleotides which are at least 80%, preferably 90%,and more preferably 95%, identical to a polynucleotide encoding a PSCFpolypeptide corresponding to amino acids 1 to 196 of SEQ ID NO:9, or acorresponding soluble PSCF polypeptide from which the membrane-bindingsegment is omitted. The above mentioned documents that relate to variousforms of PSCF are all incorporated herein by reference.

One means for isolating a polynucleotide encoding native PSCF or thePSCF according to the present invention is to utilize suchpolynucleotide or the polynucleotide according to the present invention(or one of their complements) as a probe. Thus, a natural orartificially designed probe using art recognized procedures (see, forexample: Current Protocols in Molecular Biology, Ausubel F. M. et al.(EDS.) Green Publishing Company Assoc. and John Wiley Interscience, NewYork, 1989, 1992) may be utilized. It is appreciated by one skilled inthe art that the polynucleotides of SEQ ID NO:8, or fragments thereof(comprising at least 12 contiguous nucleotides), are particularly usefulprobes. Other particularly useful probes for this purpose arehybridizable fragments of the sequences of SEQ ID NOS: 1 or 5 (i.e.,comprising at least 12 contiguous nucleotides).

With respect to nucleic acid sequences which hybridize to specificnucleic acid sequences disclosed herein, hybridization may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions. As an example of oligonucleotide hybridization, apolymer membrane containing immobilized denatured nucleic acids is firstprehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 MNaCI, 50 mM NaH₂PO₄, pH 7.0, 5.0 mM NA₂EDTA, 0.5% SDS, 10×Denhardt's,and 0.5 mg/mL polyriboadenylic acid. Approximately 2×10⁷ cpm (specificactivity 4-9×10⁸ cpm/ug) of ³²P end-labeled oligonucleotide probe arethen added to the solution. After 12-16 hours of incubation, themembrane is washed for 30 minutes at room temperature in 1×SET (150 mMNaCI, 20 mM Tris hydrochloride, pH 7.8. 1 mM Na₂EDTA) containing 0.5%SDS, followed by a 30 minute wash in fresh 1×SET at Tm less 10° C. forthe oligo-nucleotide probe. The membrane is then exposed toauto-radiographic film for detection of hybridization signals.

Stringent conditions means hybridization will occur only if there is atleast 90% identity, preferably at least 95% identity and most preferablyat least 97% identity between the sequences. Further, it is understoodthat a section of a 100 bps sequence that is 95 bps in length has 95%identity with the 1090 bps sequence from which it is obtained. See J.Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory (1989) which is hereby incorporated byreference in its entirety. Also, it is understood that a fragment of a100 bps sequence that is 95 bps in length has 95% identity with the 100bps sequence from which it is obtained.

As used herein, a first DNA (RNA) sequence is at least 70% andpreferably at least 80%, and more preferably at least a 90%, and evenmore preferably or at least 95% identical to another DNA (RNA) sequenceif there is at least 70% and preferably at least a 80% 90% or 95%identity, respectively, between the bases of the first sequence and thebases of the another sequence, when properly aligned with each other,for example when aligned by BLASTN.

The present invention relates to polynucleotides which differ from thereference polynucleotide in a manner such that the change or changesis/are silent change, in that the amino acid sequence encoded by thepolynucleotide remains the same. The present invention also relates tonucleotide changes which result in amino acid substitutions, additions,deletions fusions and truncations in the polypeptide encoded by thereference polynucleotide. In a preferred aspect of the invention thesepolypeptides retain the same biological action as the polypeptideencoded by the reference polynucleotide.

The polynucleotides of the present invention may be in the form of RNAor DNA which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNAmay be double-stranded or single-stranded, and if single stranded may bethe coding strand or non-coding (anti-sense) strand. The codingsequences which encode the mature polypeptides (mature polypeptide mayexclude the leader sequence and may optionally have an N-terminalmethionine group such as when produced by an E. coli host cell or otherprokaryotic host cell) and may be identical to the coding sequence shownin FIG. 4, (SEQ ID NOS:8) or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptides as does the DNA ofFIG. 4, (SEQ ID NOS:9, without the leader sequence −25 to −1, which mayor may not be replaced with an N-terminal methionine group).

The polynucleotides which encode each of the mature polypeptide (SEQ IDNOS:8 absent the leader sequence −25 to −1) may include, but each is notlimited to: only the coding sequence for the mature polypeptide; thecoding sequence for the mature polypeptide and additional codingsequence such as a leader sequence or a propolypeptide sequence; thecoding sequence for the mature polypeptide (and optionally additionalcoding sequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptides having the deduced amino acid sequencesof FIG. 4 (SEQ ID NO:9).

The variant in the non-exon 6 portion of the poly-nucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide. Of course thevariant in its portion that replaces exon 6 may be varied in any mannerwhich does not deactivate the overall polypeptide. In particular,polynucleotide sequences that encode relatively neutral polpeptides thatare not capable of two or three-dimensional cross-bonding, e.g., sulfidebonding, are preferred.

Thus, the present invention includes polynucleotides encoding the samemature polypeptides as shown in FIG. 4 (absent the leader sequence), aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIG. 4. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 4 (with regard to the non-exon 6 portions). As known inthe art, an allelic variant is an alternate form of a polynucleotidesequence which may have a substitution, deletion or addition of one ormore nucleotides, which does not substantially alter the function of theencoded polypeptide. Also, using directed and other evolutionstrategies, one may make very minor changes in DNA sequence which canresult in major changes in function.

Fragments of the full length gene of the present invention may be usedas hybridization probes for a cDNA or a genomic library to isolate thefull length DNA and to isolate other DNAs which have a high sequenceidentity to the gene. Probes of this type preferably have at least 10,preferably at least 15, and even more preferably at least 30 bases andmay contain, for example, at least 50 or more bases. In fact, probes ofthis type having at least up to 150 bases or greater may be utilized.The probe may also be used to identify a DNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides, having a sequencecomplementary to that of the gene or portion of the gene sequences ofthe present invention are used to screen a library of genomic DNA todetermine which members of the library the probe hybridizes to in acomplementary sense, have an identity as described above.

It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or polypeptides capable of catalyzingthe formation of a detectable product. The probes are thus useful toisolate complementary copies of DNA from other sources or to screen suchsources for related sequences.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. (As indicated above, 70% identity would includewithin such definition a 70 bps fragment taken from a 100 bppolynucleotide, for example.) The present invention particularly relatesto polynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptides encoded by the DNA of FIGS. 1A-D and 2A-E,respectively. In referring to identity in the case of hybridization, asknown in the art, such identity refers to complementarity ofpolynucleotide segments.

Alternatively, the polynucleotide may have at least 15 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto any part of a polynucleotide of the present invention and which hasan identity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:8, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% identity and morepreferably at least a 95% identity to a polynucleotide which encodes thepolypeptide of SEQ ID NO:9, as well as fragments thereof, whichfragments have at least 15 bases, preferably at least 30 bases, morepreferably at least 50 bases and most preferably fragments having up toat least 150 bases or greater, which fragments are at least 90%identical, preferably at least 95% identical and most preferably atleast 97% identical to any portion of a polynucleotide of the presentinvention.

The present invention further relates to polypeptides which have thededuced amino acid sequence of FIG. 4, (SEQ ID NO:9) as well asfragments, analogs and derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring to eachof the polypeptides of FIG. 4, respectively, (SEQ ID NO:9, respectively)mean a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes apropolypeptide which can be activated by cleavage of the propolypeptideportion to produce an active mature polypeptide.

Furthermore, regardless of the absence or presence of biological PSCFactivity, all of the polypeptides encoded by polynucleotides of thepresent invention having at least 70% polynucleotide sequence identityto the polynucleotide of SEQ ID NO:8 in the non-exon 6 portions,preferably at least 80% identity, more preferably at least 85%identical, even more preferably at least 90% identical, even furthermore preferably at least 95% identical and most preferably at least 97%identical. (or the complement polynucleotide) are useful as markerpolypeptides. Such polypeptides can be utilized to produce antibodiesagainst themselves (such as monoclonal antibodies) which can be utilizedto detect or isolate such polypeptides. Thus, the successfully insertionof a construct comprising such polynucleotides into a host cell can bedetected by utilizing such antibodies to assay for the presence of thepolypeptide. Also, higher producing cell lines can be thus identified.

The polypeptides of the present invention may be a recombinantpolypeptide and may comprise portions of a natural polypeptide or asynthetic polypeptide, but it is preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 4 (SEQ IDNO:9) may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a propolypeptide sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:9 (in particular the mature polypeptides) as well as polypeptideswhich in the non-exon 6 derived portions have at least 70% similarity(preferably at least 70% identity) to the polypeptide of SEQ ID NO:9,more preferably at least 80% identity, even more preferably at least 85%identity, and further more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of SEQ ID NO:9, andstill more preferably at least 95% similarity (still more preferably atleast 95% identity) to the polypeptide of SEQ ID NO:9 with respect tothe non-exon 6 derived portions, and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids andmost preferably at least up to 150 amino acids, or more. The maturepolypeptides according to the invention may comprise or omit anN-terminal methionine amino acid residue.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.The definition of 70% similarity would include a 70 amino acid sequencefragment of a 100 amino acid sequence, for example, or a 70 amino acidsequence obtained by sequentially or randomly deleting 30 amino acidsfrom the 100 amino acid sequence.

A variant, i.e. a “fragment”, “analog” or “derivative” polypeptide, andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions. fusions and truncations, which maybe present in any combination.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Most highly preferred are variants which retain the same biologicalfunction and activity as the reference polypeptide from which it varies.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof protiens of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector such as an expression vector. The vector maybe, for example, in the form of a plasmid, a phage, etc. The engineeredhost cells can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying the genes of the present invention. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan. Preferred are host cells are to whosesurface the polypeptide becomes bound or anchored.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or tip, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the polypeptide.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli. Streptomyces, Bacillus subtilis;fungal cells, such as yeast; insect cells such as Dirosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. Preferred host cells are fibroblastcells from any species, preferably murine or porcine fibroblast cells,particularly preferred fibroblast are murine fibroblast cells and evenmore preferred are cells from the murine STO cell line. The selection ofan appropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia);Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL SV40(Pharmacia). However, any other plasmid or vector may be used as long asthey are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukarvotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection. DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature polypeptides can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Depending upon the expression host a mature polypeptide may or may notcontain an N-terminal methionine. Cell-free translation systems can alsobe employed to produce such polypeptides using RNAs derived from the DNAconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor, N.Y., (1989), the disclosure of which is herebyincorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock polypeptides, among others. The heterologous structural sequenceis assembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated polypeptide. Optionally, theheterologous sequence can encode a fusion polypeptide including anN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired polypeptide together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of polypeptides can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant polypeptide. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptides according to the invention can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Polypeptide refolding stepscan be used, as necessary, in completing configuration of the maturepolypeptide. Finally, high performance liquid chromatography (HPLC) canbe employed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Antibodies generated against a polypeptide corresponding to a sequenceof the present invention can be obtained by direct injection of therespective polypeptide (or a portion of the polypeptide) into an animalor by administering the polypeptides to an animal, preferably anon-human. The antibody so obtained will then bind the respectivepolypeptide itself In this manner, even a sequence encoding only afragment of the polypeptides can be used to generate antibodies bindingthe whole native polypeptides. Such antibodies can then be used toisolate the polypeptide from cells expressing that polypeptide and mayalso be useful as antimicrobials, or controls in assays to determine theefficacy of potential antimicrobials.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express porcine antibodies to immunogenicpolypeptide products of this invention.

Antibodies generated against a polypeptide of the present invention maybe used in screening for similar polypeptides from other organisms andsamples. Such screening techniques are known in the art, for example,one such screening assay is described in Sambrook and Maniatis.Molecular Cloning: A Laboratory Manual (2d Ed.), vol. 2: Section 8.49,Cold Spring Harbor Laboratory, 1989, which is hereby incorporated byreference in its entirety. Further, such antibodies are useful to detectthe successful insertion of a transcript comprising the polynucleotideencoding the polypeptide which can be bound by such antibodies. Thus,the antibodies can be utilized to detect the “marker” polypeptide thatis encoded by the polynucleotide inserted into the host cell.

The following non-limiting examples are provided merely to illustrate apreferred embodiment of the invention.

EXAMPLE 1

Preparation of STO8 Feeder Cells or Similar Feeder Cell Lines

A membrane-bound form of porcine stem cell factor is obtained byutilizing the STO cell line, which is a thioguanine/oubain resistantsub-line of SIM mouse fibroblasts, Virology 50:339 (1972); STO cells asdescribed in U.S. Pat. No. 5,453,357). The STO cells are transfectedwith a membrane-bound portion which encodes an active PSCF polypeptide.The full-length sequence for PSCF (SEQ ID NO:2) based on native cDNA(SEQ ID NO:1) is reported in Biology of Reproduction 50: 95-102 (1994)(see also, FIG. 1. attached hereto), and an active form is produced bytransfecting STO cells with active fragments of the cDNA coding sequencefrom which the polynucleotides encoding the leader sequence amino acids(−25 to −1) are removed and exon 6 polynucleotides are replaced with atri-nucleotide fragment encoding the amino acid “Gly”.

The following general procedure is followed. RNA is isolated from pigbone marrow stromal cells, RT-PCR is performed utilizing 1 μg total RNAin 6 μl H₂O which is incubated at 65° C. for 3 minutes, then chilled onice. Then added is 4 μl 5xRT buffer, 2 μl 0.1 M Dithiothreitol (DTT), 1μl RNasin (Promega, Madison, Wis.), 2 μl 30 μM oligo dT₁₆, 2 μl dNTPs(10 mM each dATP, dTTP, dGTP, dCTP), 2 μl mg/ml BSA, 1 μl reversetranscriptase (Gibco Life Technologies, Baltimore, MD) and the reactionmixture is incubated at room temperature for 10 min., 42° C. for 60 min.90° C. for 5 min. Then added is 1 μ RNase H (4 units, Gibco LifeTechnologies, Baltimore, Md.) and the reaction is incubated at 37° C.for 20 min, prior to Sephadex™ G-50 column chromatography to purify thecDNA product. The cDNA product is subjected to PCR using theoligonucleotides 5′MSFHindIII (5′GGT CAA GCT TCG CTG CCT TTC CTT ATG AAGAAG, SEQ ID NO: 3) and 3′MSFXbaI (5TCC ATC TAG AAC CAC CCA ATG TAC GAAAGC AAC, SEQ ID. NO: 4). SEQ ID NO: 1 contains a HindIII site andincludes nucleotides 1-24 of SEQ ID NO: 3 (SEQ ID. NO:5 in thisapplication) of U.S. Pat. No. 5,589,582). SEQ ID. NO: 4 contains an XbaIsite and the reverse complement of nucleotides 915 through 935 ofLO7786. The resulting PCR product is cleaved with HindIII and XbaI andcloned in pRcCMV (Invitrogen, Portland, Oreg.). The resulting plasmid isdescribed as pSCFpRcCMV#2 and contains the full-length porcine cDNA forstem cell factor.

Xba and StuI are used to cleave pSCFpRcCMV#2 and a DNA fragment ofapproximately 250 bp (fragment 1) is isolated. ClaI and XbaI are alsoused to cleave pSCFpRcCMV and a DNA fragment of approximately 6.2 Kb isisolated (fragment 2). Two oligonucleotides described as 5′SCFlk (SEQ IDNO: 6, ATCCATCGAT GCCTTCAAGG ATTTGGAGAT GGTGGCACCT AAAACTAGTG AATGTGTGATTTCTTCAA) and 3′SCFlk (SEQ ID NO: 7 TCT GAGGCCTTCC TATTACTCT ACTGCTGTCATTCCCTI=CAGGAGTTAA TGTTGAAGAA ATC) are synthesized. The oligonucleotides(1 μg (10 μg) of SEQ ID NO: 6 and 1 μg) 10 μl) SEQ ID NO: 7 are mixedwith 3 μl 10×Klenow butter [SambrooK, 1989#1973] and incubated at 75° C.for 5 min, and then allowed to cool slowly. Afterwards 2 μl 2.5 mM eachdXTP, 1.5 μl (7.5 unit) DNA polymerase Klenow fragment 3.5 μl H20 areadded. After 30 min at 37° C., the reaction is heated at 70° C. for 10min. The DNA fragment (fragment 3) is cleaved with ClaI and StuI. Athree-way ligation is then performed with the DNA fragments 1,2 and 3. Aresulting plasmid pPSCF is identified to have the correct sequence,shown in FIG. 4 (SEQ ID NO: 8 encoding amino acid sequence SEQ ID. NO:9). The plasmid does not contain exon 6 and therefore is a form of SCFthat is ordinarily expressed preferentially as a membrane bound form.

STO cells are electroporated according to the BIORAD (Hercules, Calif.)instructions for use of the Gene Pulses® Electroprotocols, using PvuIlinerized pPSCF. Cells are selected for growth in G418 (500 μg/ml) andanalyzed for the expression of the modified PSCF, using RT-PCR from RNAisolated from G418 resistant clones. Examples of STO cell lines that aresuccessfully transfected with the polynucleotides of the above plasmidare designated as cell lines STO5, STO8, STO12 and STO18.

Cells (STO (control expressing murine membrane SCF), STO8, STO12 andSTO18) are plated into 96 well flat bottom plates in Iscove's ModifiedDulbecco's Media containing 10% heat-inactivated fetal bovine serum.Prior to the addition of bone marrow cells, the plates are irradiated toprevent further proliferation of the STO, STO5. STO8, STO12, and STO18cells. Bone marrow cells are added to the wells. After 2 days inculture, 1 microcurie of ³H-Tdr is added to each well, and the platesare harvested on day 3. Results are counts per minute (cpm) andexpressed as a mean value of triplicate plates. FIG. 3 shows that eachof the transfected STO cell lines supports the proliferation of pig bonemarrow cells to a greater extent than the untransfected STO cell line.The proliferative response on the bone marrow cells of the transfectedcells is similar to that observed with untransfected STO cells that werecultured in combination with of 100-200 μg soluble pig SCF (for example,as set forth in U.S. Pat. No. 5,589,582). (data not reported)

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

11 952 BASE PAIRS NUCLEIC ACID SINGLE LINEAR cDNA 1 GAGCTCCAGAACAGGTAAAC GGAGTTGCCA CACCGCTGCC TGGGCTGGAT CACAGCGCTG 60 CCTTTCCTT ATGAAG AAG ACA CAA ACT TGG ATT ATC ACT TGC ATT TAT 108 Met Lys Lys Thr GlnThr Trp Ile Ile Thr Cys Ile Tyr -25 -20 -15 CTT CAA CTG CTC CTA TTT AATCCT CTC GTC AGA ACT CAA GGG ATC TGC 156 Leu Gln Leu Leu Leu Phe Asn ProLeu Val Arg Thr Gln Gly Ile Cys -10 -5 1 AGG AAC CGT GTG ACT GAT GAT GTGAAA GAC GTT ACA AAA TTG GTG GCA 204 Arg Asn Arg Val Thr Asp Asp Val LysAsp Val Thr Lys Leu Val Ala 5 10 15 20 AAT CTT CCA AAA GAC TAT AAG ATAACC CTC AAA TAT GTC CCC GGG ATG 252 Asn Leu Pro Lys Asp Tyr Lys Ile ThrLeu Lys Tyr Val Pro Gly Met 25 30 35 GAC GTT TTG CCT AGT CAT TGT TGG ATAAGC GAA ATG GTG GAA CAA CTG 300 Asp Val Leu Pro Ser His Cys Trp Ile SerGlu Met Val Glu Gln Leu 40 45 50 TCA GTC AGC TTG ACT GAT CTT CTG GAC AAGTTT TCC AAT ATT TCT GAA 348 Ser Val Ser Leu Thr Asp Leu Leu Asp Lys PheSer Asn Ile Ser Glu 55 60 65 GGC TTG AGT AAT TAT TCT ATC ATA GAC AAA CTTGTG AAA ATT GTT GAT 396 Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu ValLys Ile Val Asp 70 75 80 GAC CTC GTG GAA TGC ATG GAA GAA CAC TCA TTT GAGAAT GTA AGA AAA 444 Asp Leu Val Glu Cys Met Glu Glu His Ser Phe Glu AsnVal Arg Lys 85 90 95 100 TCA TCT AAG AGC CCA GAA CCC AGG CTG TTT ACT CCTGAA AAA TTC TTT 492 Ser Ser Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro GluLys Phe Phe 105 110 115 GGG ATT TTT AAT AGA TCC ATC GAT GCC TTC AAG GATTTG GAG ATG GTG 540 Gly Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp LeuGlu Met Val 120 125 130 GCA CCT AAA ACT AGT GAA TGT GTG ATT TCT TCA ACATTA ACT CCT GAA 588 Ala Pro Lys Thr Ser Glu Cys Val Ile Ser Ser Thr LeuThr Pro Glu 135 140 145 AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATGTTA CCC CCT GTT 636 Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met LeuPro Pro Val 150 155 160 GCA GCC AGC TCC CTT AGG AAT GAC AGC AGT AGC AGTAAT AGG AAA GCC 684 Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser AsnArg Lys Ala 165 170 175 180 TCA GAT TCG ATT GAA GAC TCC AGC CTC CAG TGGGCA GCG GTA GCA TTG 732 Ser Asp Ser Ile Glu Asp Ser Ser Leu Gln Trp AlaAla Val Ala Leu 185 190 195 CCA GCA TTC TTC TCT CTT GTG ATT GGG TTT GCTTTT GGA GCC TTA TAC 780 Pro Ala Phe Phe Ser Leu Val Ile Gly Phe Ala PheGly Ala Leu Tyr 200 205 210 TGG AAG AAG AAA CAA CCA AAC CTT ACA AGG ACAGTG GAA AAT ATA GAG 828 Trp Lys Lys Lys Gln Pro Asn Leu Thr Arg Thr ValGlu Asn Ile Gln 215 220 225 ATT AAT GAA GAG GAT AAT GAG ATA AGT ATG TTGCAA GAA AAA GAG AGA 876 Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu GlnGlu Lys Glu Arg 230 235 240 GAG TTT CAA GAA GTG TAA TTGTGGCGTGTATCAACACT GTTGCTTTCG TACATTGGT 934 Glu Phe Gln Glu Val 245 GGTAACAGTTGATGTTTG 952 274 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 2 MetLys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile Tyr Leu Gln -25 -20 -15 LeuLeu Leu Phe Asn Pro Leu Val Arg Thr Gln Gly Ile Cys Arg -10 -5 1 5 AsnArg Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala 10 15 20 Asn LeuPro Lys Asp Tyr Lys Ile Thr Leu Lys Tyr Val Pro Gly 25 30 35 Met Asp ValLeu Pro Ser His Cys Trp Ile Ser Glu Met Val Glu 40 45 50 Gln Leu Ser ValSer Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn 55 60 65 Ile Ser Glu Gly LeuSer Asn Tyr Ser Ile Ile Asp Lys Leu Val 70 75 80 Lys Ile Val Asp Asp LeuVal Glu Cys Met Glu Glu His Ser Phe 85 90 95 Glu Asn Val Arg Lys Ser SerLys Ser Pro Glu Pro Arg Leu Phe 100 105 110 Thr Pro Glu Lys Phe Phe GlyIle Phe Asn Arg Ser Ile Asp Ala 115 120 125 Phe Lys Asp Leu Glu Met ValAla Pro Lys Thr Ser Glu Cys Val 130 135 140 Ile Ser Ser Thr Leu Thr ProGlu Lys Asp Ser Arg Val Ser Val 145 150 155 Thr Lys Pro Phe Met Leu ProPro Val Ala Ala Ser Ser Leu Arg 160 165 170 Asn Asp Ser Ser Ser Ser AsnArg Lys Ala Ser Asp Ser Ile Glu 175 180 185 Asp Ser Ser Leu Gln Trp AlaAla Val Ala Leu Pro Ala Phe Phe 190 195 200 Ser Leu Val Ile Gly Phe AlaPhe Gly Ala Leu Tyr Trp Lys Lys 205 210 215 Lys Gln Pro Asn Leu Thr ArgThr Val Glu Asn Ile Gln Ile Asn 220 225 230 Glu Glu Asp Asn Glu Ile SerMet Leu Gln Glu Lys Glu Arg Glu 235 240 245 Phe Gln Glu Val 33 BASEPAIRS NUCLEIC ACID SINGLE LINEAR DNA 3 GGTCAAGCTT CGCTGCCTTT CCTTATGAAGAAG 33 33 BASE PAIRS NUCLEIC ACID SINGLE LINEAR DNA 4 TCCATCTAGAACCACCCAAT GTACGAAAGC AAC 33 633 BASE PAIRS NUCLEIC ACID SINGLE LINEARcDNA 5 GCGCT GCCTTTCCTT 15 ATG AAG AAG ACA CAA ACT TGG ATT ATC ACT TGCATT TAT CTT CAA CTG 63 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys IleTyr Leu Gln Leu -25 -20 -15 -10 CTC CTA TTT AAT CCT CTC GTC AGA ACT CAAGGG ATC TGC AGG AAC CGT 111 Leu Leu Phe Asn Pro Leu Val Arg Thr Gln GlyIle Cys Arg Asn Arg -5 1 5 GTG ACT GAT GAT GTG AAA GAC GTT ACA AAA TTGGTG GCA AAT CTT CCA 159 Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu ValAla Asn Leu Pro 10 15 20 AAA GAC TAT AAG ATA ACC CTC AAA TAT GTC CCC GGGATG GAC GTT TTG 207 Lys Asp Tyr Lys Ile Thr Leu Lys Tyr Val Pro Gly MetAsp Val Leu 25 30 35 CCT AGT CAT TGT TGG ATA AGC GAA ATG GTG GAA CAA CTGTCA GTC AGC 255 Pro Ser His Cys Trp Ile Ser Glu Met Val Glu Gln Leu SerVal Ser 40 45 50 55 TTG ACT GAT CTT CTG GAC AAG TTT TCC AAT ATT TCT GAAGGC TTG AGT 303 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu GlyLeu Ser 60 65 70 AAT TAT TCT ATC ATA GAC AAA CTT GTG AAA ATT GTT GAT GACCTC GTG 351 Asn Tyr Ser Ile Ile Asp Lys Leu Val Lys Ile Val Asp Asp LeuVal 75 80 85 GAA TGC ATG GAA GAA CAC TCA TTT GAG AAT GTA AGA AAA TCA TCTAAG 399 Glu Cys Met Glu Glu His Ser Phe Glu Asn Val Arg Lys Ser Ser Lys90 95 100 AGC CCA GAA CCC AGG CTG TTT ACT CCT GAA AAA TTC TTT GGG ATTTTT 447 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Lys Phe Phe Gly Ile Phe105 110 115 AAT AGA TCC ATC GAT GCC TTC AAG GAT TTG GAG ATG GTG GCA CCTAAA 495 Asn Arg Ser Ile Asp Ala Phe Lys Asp Leu Glu Met Val Ala Pro Lys120 125 130 135 ACT AGT GAA TGT GTG ATT TCT TCA ACA TTA ACT CCT GAA AAAGAT TCC 543 Thr Ser Glu Cys Val Ile Ser Ser Thr Leu Thr Pro Glu Lys AspSer 140 145 150 AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA CCC CCT GTT GCAGCC AGC 591 Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala AlaSer 155 160 165 TCC CTT AGG AAT GAC AGC AGT AGC AGT AAT AGG AAA GCC TAA633 Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Arg Lys Ala 170 175 180 68BASE PAIRS NUCLEIC ACID SINGLE LINEAR DNA 6 ATCCATCGAT GCCTTCAAGGATTTGGAGAT GGTGGCACCT AAAACTAGTG AATGTGTGAT 60 TTCTTCAA 68 65 BASE PAIRSNUCLEIC ACID SINGLE LINEAR DNA 7 TCTGAGGCCT TCCTATTACT CTACTGCTGTCATTCCCTTT TTCAGGAGTT AATGTTGAAG 60 AAATC 65 828 BASE PAIRS NUCLEIC ACIDSINGLE LINEAR cDNA 8 CGCTGCCTTT CCTT ATG AAG AAG ACA CAA ACT TGG ATT ATCACT TGC ATT 50 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile -25 -20-15 TAT CTT CAA CTG CTC CTA TTT AAT CCT CTC GTC AGA ACT CAA GGG ATC 98Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Arg Thr Gln Gly Ile -10 -5 1TGC AGG AAC CGT GTG ACT GAT GAT GTG AAA GAC GTT ACA AAA TTG GTG 146 CysArg Asn Arg Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val 5 10 15 GCAAAT CTT CCA AAA GAC TAT AAG ATA ACC CTC AAA TAT GTC CCC GGG 194 Ala AsnLeu Pro Lys Asp Tyr Lys Ile Thr Leu Lys Tyr Val Pro Gly 20 25 30 35 ATGGAC GTT TTG CCT AGT CAT TGT TGG ATA AGC GAA ATG GTG GAA CAA 242 Met AspVal Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Glu Gln 40 45 50 CTG TCAGTC AGC TTG ACT GAT CTT CTG GAC AAG TTT TCC AAT ATT TCT 290 Leu Ser ValSer Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser 55 60 65 GAA GGC TTGAGT AAT TAT TCT ATC ATA GAC AAA CTT GTG AAA ATT GTT 338 Glu Gly Leu SerAsn Tyr Ser Ile Ile Asp Lys Leu Val Lys Ile Val 70 75 80 GAT GAC CTC GTGGAA TGC ATG GAA GAA CAC TCA TTT GAG AAT GTA AGA 386 Asp Asp Leu Val GluCys Met Glu Glu His Ser Phe Glu Asn Val Arg 85 90 95 AAA TCA TCT AAG AGCCCA GAA CCC AGG CTG TTT ACT CCT GAA AAA TTC 434 Lys Ser Ser Lys Ser ProGlu Pro Arg Leu Phe Thr Pro Glu Lys Phe 100 105 110 115 TTT GGG ATT TTTAAT AGA TCC ATC GAT GCC TTC AAG GAT TTG GAG ATG 482 Phe Gly Ile Phe AsnArg Ser Ile Asp Ala Phe Lys Asp Leu Glu Met 120 125 130 GTG GCA CCT AAAACT AGT GAA TGT GTG ATT TCT TCA ACA TTA ACT CCT 530 Val Ala Pro Lys ThrSer Glu Cys Val Ile Ser Ser Thr Leu Thr Pro 135 140 145 GAA AAA GGG AATGAC AGC AGT AGC AGT AAT AGG AAA GCC TCA GAT TCG 578 Glu Lys Gly Asn AspSer Ser Ser Ser Asn Arg Lys Ala Ser Asp Ser 150 155 160 ATT GAA GAC TCCAGC CTC CAG TGG GCA GCG GTA GCA TTG CCA GCA TTC 626 Ile Glu Asp Ser SerLeu Gln Trp Ala Ala Val Ala Leu Pro Ala Phe 165 170 175 TTC TCT CTT GTGATT GGG TTT GCT TTT GGA GCC TTA TAC TGG AAG AAG 674 Phe Ser Leu Val IleGly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys 180 185 190 195 AAA CAA CCAAAC CTT ACA AGG ACA GTG GAA AAT ATA GAG ATT AAT GAA 722 Lys Gln Pro AsnLeu Thr Arg Thr Val Glu Asn Ile Gln Ile Asn Glu 200 205 210 GAG GAT AATGAG ATA AGT ATG TTG CAA GAA AAA GAG AGA GAG TTT CAA 770 Glu Asp Asn GluIle Ser Met Leu Gln Glu Lys Glu Arg Glu Phe Gln 215 220 225 GAA GTG TAATTGTGGCGTG TATCAACACT GTTGCTTTCG TACATTGGGT GGTTCTAGA 828 Glu Val 254AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN 9 Met Lys Lys Thr GlnThr Trp Ile Ile Thr Cys Ile Tyr Leu Gln -25 -20 -15 Leu Leu Leu Phe AsnPro Leu Val Arg Thr Gln Gly Ile Cys Arg -10 -5 1 5 Asn Arg Val Thr AspAsp Val Lys Asp Val Thr Lys Leu Val Ala 10 15 20 Asn Leu Pro Lys Asp TyrLys Ile Thr Leu Lys Tyr Val Pro Gly 25 30 35 Met Asp Val Leu Pro Ser HisCys Trp Ile Ser Glu Met Val Glu 40 45 50 Gln Leu Ser Val Ser Leu Thr AspLeu Leu Asp Lys Phe Ser Asn 55 60 65 Ile Ser Glu Gly Leu Ser Asn Tyr SerIle Ile Asp Lys Leu Val 70 75 80 Lys Ile Val Asp Asp Leu Val Glu Cys MetGlu Glu His Ser Phe 85 90 95 Glu Asn Val Arg Lys Ser Ser Lys Ser Pro GluPro Arg Leu Phe 100 105 110 Thr Pro Glu Lys Phe Phe Gly Ile Phe Asn ArgSer Ile Asp Ala 115 120 125 Phe Lys Asp Leu Glu Met Val Ala Pro Lys ThrSer Glu Cys Val 130 135 140 Ile Ser Ser Thr Leu Thr Pro Glu Lys Gly AsnAsp Ser Ser Ser 145 150 155 Ser Asn Arg Lys Ala Ser Asp Ser Ile Glu AspSer Ser Leu Gln 160 165 170 Trp Ala Ala Val Ala Leu Pro Ala Phe Phe SerLeu Val Ile Gly 175 180 185 Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys LysGln Pro Asn Leu 190 195 200 Thr Arg Thr Val Glu Asn Ile Gln Ile Asn GluGlu Asp Asn Glu 205 210 215 Ile Ser Met Leu Gln Glu Lys Glu Arg Glu PheGln Glu Val 220 225 205 AMINO ACIDS AMINO ACID <Unknown> LINEAR PROTEIN10 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile Tyr Leu Gln Leu -25-20 -15 -10 Leu Leu Phe Asn Pro Leu Val Arg Thr Gln Gly Ile Cys Arg AsnArg -5 1 5 Val Thr Asp Asp Val Lys Asp Val Thr Lys Leu Val Ala Asn LeuPro 10 15 20 Lys Asp Tyr Lys Ile Thr Leu Lys Tyr Val Pro Gly Met Asp ValLeu 25 30 35 Pro Ser His Cys Trp Ile Ser Glu Met Val Glu Gln Leu Ser ValSer 40 45 50 55 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu GlyLeu Ser 60 65 70 Asn Tyr Ser Ile Ile Asp Lys Leu Val Lys Ile Val Asp AspLeu Val 75 80 85 Glu Cys Met Glu Glu His Ser Phe Glu Asn Val Arg Lys SerSer Lys 90 95 100 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Lys Phe PheGly Ile Phe 105 110 115 Asn Arg Ser Ile Asp Ala Phe Lys Asp Leu Glu MetVal Ala Pro Lys 120 125 130 135 Thr Ser Glu Cys Val Ile Ser Ser Thr LeuThr Pro Glu Lys Asp Ser 140 145 150 Arg Val Ser Val Thr Lys Pro Phe MetLeu Pro Pro Val Ala Ala Ser 155 160 165 Ser Leu Arg Asn Asp Ser Ser SerSer Asn Arg Lys Ala 170 175 180 6 BASE PAIRS NUCLEIC ACID SINGLE LINEARDNA 11 TCTAGA 6

What is claimed is:
 1. A recombinant vector comprising a polynucleotidewherein said polynucleotide is DNA and said polynucleotide is selectedfrom the group consisting of (a) a polynucleotide encoding a polypeptidecomprising amino acids 1 to 196 of SEQ ID NO:9 and (b) the complement of(a).
 2. A recombinant host cell comprising a polynucleotide wherein saidpolynucleotide is a DNA and said polynucleotide is selected from thegroup consisting of (a) a polynucleotide encoding a polypeptidecomprising amino acids 1 to 196 of SEQ ID NO:9 and (b) the complement of(a).
 3. A recombinant host cell according to claim 2, wherein said hostcell is a murine or porcine fibroblast cell.
 4. The recombinant hostcell according to claim 3, wherein said host cell is a murine STO cell.5. A recombinant host cell according to claim 2, wherein said host cellis an STO cell.
 6. A recombinant host cell according to claim 2, whereinsaid host cell will produce the polypeptide encoded by saidpolynucleotide as a polypeptide anchored to its surface.
 7. A method forproducing a polypeptide comprising expressing from the recombinant cellof claim 2 the polypeptide encoded by said polynucleotide.